Method of stabilizing lipid-rich plaque and method of preventing rupture thereof

ABSTRACT

A method of stabilizing lipid-rich plaques and method of preventing the rupture thereof, characterized in that an effective amount of 2-[4-[2-(benzimidazol-2-ylthio)ethyl]piperazin-1-yl]-N-[2,4-bis(methylthio)-6-methyl-3-pyridyl]acetamide, its acid adduct salt or a hydrate thereof is administered to patients with lipid-rich plaques. Prevention of plaque rupture and stabilization of plaques can be attained by reducing the occupancy of macrophages in lipid-rich plaques being unstable and tending to rupture among plaques being a lesion from a seat of atherosclerosis and simultaneously increasing the occupancy of collagen therein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/569,642, filed on Feb. 27, 2006, which is a 371 National Stage ofInternational Application No. PCT/JP2004/011935, filed Aug. 19, 2004,which is based upon and claims the benefit of priority from the priorU.S. Application No. 60/498,610, filed on Aug. 29, 2003, the entirecontents of all of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a method of stabilizing lipid-richplaques in the atherosclerotic lesions and a method of preventing therupture thereof. More specifically, the present invention relates to amethod of stabilizing lipid-rich plaques and a method of preventing therupture thereof by suppressing accumulation of macrophages andincreasing collagen in the plaque lesions by administering an effectiveamount of2-[4-[2-(benzimidazol-2-ylthio)ethyl]piperazin-1-yl]-N-[2,4-bis(methylthio)-6-methyl-3-pyridyl]acetamide,an acid salt thereof or a hydrate thereof.

BACKGROUND ART

Recently, arteriosclerotic diseases are increasing due to changes oflife-style as a result of improvement in living standards such asingestion of a high-calorie diet and a high-cholesterol diet, lack ofexercise, obesity, stress caused by social complexities, aging society,and the like. Risk factors of arteriosclerotic diseases can beclassified into smoking, obesity, hypertension, hyperuricemia, diabetesmellitus, hyperlipidemia, and the like. Among them, hyperlipidemia suchas hypertriglyceridemia, hypo-HDL (HDL: high-density lipoprotein) andhyper-LDL (LDL: low-density lipoprotein) draws increasing attention, andparticularly lowering hyper-cholesterol level is placed emphasis as anobject for the pharmacotherapy, and various treatments have beenperformed. In particular, a drug (statin drug), which can achievesignificant results in treatment of hypercholesterolemia, is the druginhibiting HMG-CoA (HMG-CoA: 3-hydroxy-3-methylglutaryl-coenzyme A)reductase, a rate-limiting enzyme of cholesterol biosynthesis, can bementioned. Cholesterol lowering therapy using statin shows successfulresults specifically in various arteriosclerotic diseases, for examplemyocardial infarction and cerebral infarction, caused by hyperlipidemia.With regard to coronary arterial diseases including acute myocardialinfarction, results of multicenter epidemiological surveys such as 4S(Scandinavian Simvastatin Survival Study) and WOS (COPS) (West ofScotland Coronary Prevention Study) were reported, and effectiveness ofsimvastatin therapy has been proven in an improvement of survival ratefor five years. However, even if the statin drug represented bysimvastatin (Patent Reference 1) and pravastatin (Patent Reference 2) issaid to be effective, improvement rate for rate of crisis of coronarydisease event is no more than about 30%, and is not satisfactorycondition in the medical field. As a mechanism for development of drugefficacy with the statin, it is known that as a result of generating theinhibition of cholesterol biosynthesis in vivo and simultaneouslyoccurring increased expression of LDL receptors accompanied by loweringcholesterol level in the liver, the increased LDL receptors promoteincorporation of blood LDL and cause to decrease total cholesterol levelin plasma. Consequently, there is a problem that complete lowering LDLcholesterol cannot be expected in patients with homozygote andheterozygote who are deficient of LDL receptor such as familialhypercholesterolemia. It is known that combined medication of fibrateand statin to the patient with hypertriglyceridemia causesrhabdomyolysis, and in case of cerivastatin (Patent Reference 3), severeside effect caused by such the medication resulted to discontinuation ofsales. Considering such background, drugs, which do not exhibitanti-arteriosclerotic action coupled with lowering total cholesterollevel in plasma but exhibit direct action to arteriosclerosis lesion, isattractive and to be expected.

Plaques, which are the primary focus of atherosclerosis, consist of thelipid core filled with cholesterol and ester thereof and the fibrousmaterial called extracellular matrix. Among the plaques, lipid-richplaques, which are predominantly made up of lipid and inflammatory cellssuch as macrophage and covered with thin fibrous membrane, are called“unstable plaques”. It is easy to rupture, and when the plaques rupture,contents of the plaques are exposed to the blood flow to promotethrombogenesis. As a result, acute coronary syndrome (ACS) such asunstable angina, acute myocardial infarction and ischemic sudden deathwill occur (Non-Patent Reference 1). Actually, it is known that as aresult of examination of culprit lesion in the cases of death caused byACS, about 75% of the cases were due to thrombogenesis accompanied byrupture of the plaques (Non-Patent Reference 2). Focusing attention onstenotic rate of blood vessel, i.e. plaque size, the culprit lesion ofmyocardial infarction was found mainly to be the region of blood vesselwith the stenotic rate below 50% (Non-Patent Reference 2). This factsuggests that causes for plaque rupture are not the size of the plaquesize but the quality of the plaque.

Involvement of matrix metalloprotease (MMP) secreted by the large amountof accumulated macrophage is considered to be direct cause for theplaque rupture, and MMP may degrade the fibrous collagen to thin andweaken the fibrous film. Further, the macrophage is reported tostimulate the thrombogenesis at the ruptured region through expressionof the tissue factor (Non-Patent Reference 3).

Consequently, prevention of the rupture of “unstable plaques” may beimportant in countermeasure for ACS. For that purpose, methods includinginhibition of the function or the accumulation of macrophages, orinhibition of the degradation of the fibrous collagen or strengtheningthe fibrous cap by increasing the collagen content may be considered. Inconclusion for prevention and treatment of acute coronary syndrome,although regression of the plaques accompanied by reducing totalcholesterol level in plasma may be effective, instead drugs forinhibiting accumulation of the macrophage and increasing the collagen,as a result, stabilizing lipid-rich plaques may be more preferable.

Considering these situations, acyl-CoA: cholesterol O-acyltransferase(ACAT) inhibitor has been drawn attention as cholesterol lowering drughaving different mechanism of action to the statin.

Although there are large numbers of reports indicating regression ofplaques by using ACAT inhibitor, significant decrease in plasma totalcholesterol level is accompanied in any cases. Consequently, it isunknown whether direct regression of plaques could be achieved or not,and as a result, there may be discrepancy in interpretation of data(Non-Patent References 4 and 5).

However, recently, several ACAT inhibitors, which show regression effectof plaques without affecting the plasma total cholesterol level, havereported. For example, JP-A-2002-255808 (Patent Reference 4) disclosesACAT inhibitor as a plaque regressing agent. WO 01/034127 (PatentReference 5) discloses ACAT inhibitor, which leads to decreaseaccumulation of macrophage and expression of MMP in the plaques.However, although the plaque reducing activities were indicated in thesereports, they fail to mention increase or decrease of the collagen, oneof important factor contributing to the stability of the plaques(Non-Patent Reference 6). Although the compound (avasimibe, hereinafterdesignated as CI-1011) examined therein exhibits plaque regressingactivity, no finding such as increase of collagen and stability isindicated.

As obvious from above, ACAT inhibitor, which is different fromconventional ACAT inhibitors, and independently reduces the occupationrate of macrophages in the plaques without affecting the plasma totalcholesterol level and has action to increase the occupation rate ofcollagen as well as preventing rupture of the plaques without leading todiffuse macrophages even if they are ruptured. Such ACAT inhibition hasnot been known.

-   Patent Reference 1: U.S. Pat. No. 4,444,784.-   Patent Reference 2: U.S. Pat. No. 4,346,227.-   Patent Reference 3: U.S. Pat. No. 5,177,080.-   Patent Reference 4: JP-A-2002-255808.-   Patent Reference 5: WO 01-034127.-   Non-Patent Reference 1: N. Engl. J. Med. 326(4): 242-50, 1992.-   Non-Patent Reference 2: Circulation 92(3): 657-71, 1995.-   Non-Patent Reference 3: Ann. N.Y. Acad. Sci. 902:140-52, 2000.-   Non-Patent Reference 4: Exp. Opin. Invest. Drugs 4: 353-387, 1995.-   Non-Patent Reference 5: Drug Discovery Today 3: 19-25, 1998.-   Non-Patent Reference 6: Circ. Res. 86: 101-8, 2000.

DISCLOSURE OF THE INVENTION Problem to be Solved by the Invention

In consideration of the above, the inventors have, after extensivelystudied, found that2-[4-[2-(benzimidazol-2-ylthio)ethyl]piperazin-1-yl]-N-[2,4-bis(methylthio)-6-methyl-3-pyridyl]acetamide(hereinafter designated as compound 1), an acid salt thereof or ahydrate thereof disclosed in Example of WO 98/054153 could reduce theoccupation rate of macrophages in the plaques and increase theoccupation rate of collagen without affecting significant changes of theplasma total cholesterol level, namely suggesting stability activity oflipid-rich plaques, and completed the present invention. ACAT inhibitor,which has selective activity for macrophage, of the compound 1 andproduction method thereof are disclosed in WO 98/054153, the descriptionof which has been incorporated herein by reference. Use of the compounddisclosed therein is treatment of hypercholesterolemia andatherosclerosis. Although it describes inhibitor for (selective) foamcell formation of macrophage, no description on the plaque stabilizingactivity is found.

Means to Solve the Problems

The present invention provides a method of stabilizing lipid-richplaques in atherosclerotic lesions and method of preventing the rupturethereof, characterized in that an effective amount of the compound 1, anacid salt thereof or a hydrate thereof is administered to a patient withlipid-rich plaques. Further, the present invention provides astabilizing agent for lipid-rich plaques and a preventing agent forrupture thereof comprising the compound 1, an acid salt thereof or ahydrate thereof as an active ingredient. Further the present inventionprovides a pharmaceutical composition having activities for stabilizinglipid-rich plaques comprising the compound 1, an acid salt or a hydratethereof, and a pharmaceutically acceptable carrier thereof. Stillfurther the present invention provides use of the compound 1, an acidsalt thereof or a hydrate thereof for production of the stabilizingagent for lipid-rich plaques and the preventing agent for rupturethereof.

The present invention provides a method of preventing thrombogenesisaccompanied to the rupture of lipid-rich plaques, characterized in thatan effective amount of the compound 1, an acid salt thereof or a hydratethereof is administered to a patient with lipid-rich plaques. Further,the present invention provides an agent having activity for preventingthrombogenesis accompanied to the rupture of lipid-rich plaquescomprising the compound 1, an acid salt thereof or a hydrate thereof asan active ingredient. Further the present invention provides apharmaceutical composition having activity for preventing thrombogenesisaccompanied to the rupture of lipid-rich plaques comprising the compound1, an acid salt thereof or a hydrate thereof, and a pharmaceuticallyacceptable carrier thereof. Still further the present invention providesuse of the compound 1, an acid salt thereof or a hydrate thereof forproduction of the pharmaceutical preparation for prevention ofthrombogenesis accompanied to the rupture of lipid-rich plaques.

The present invention provides a method of preventing and/or treatingacute coronary syndrome, characterized in that an effective amount ofthe compound 1, an acid salt thereof or a hydrate thereof isadministered to a patient with lipid-rich plaques. Further, the presentinvention provides an agent for preventing and/or treating acutecoronary syndrome comprising the compound 1, an acid salt thereof or ahydrate thereof as an active ingredient. Further the present inventionprovides a pharmaceutical composition having activities for preventingand/or treating acute coronary syndrome comprising the compound 1, anacid salt thereof or a hydrate thereof, and a pharmaceuticallyacceptable carrier thereof. Still further the present invention providesuse of the compound 1, an acid salt thereof or a hydrate thereof forproduction of the pharmaceutical preparation for preventing and/ortreating acute coronary syndrome.

The present invention provides a method of preventing and/or treatingacute myocardial infarction, characterized in that an effective amountof the compound 1, an acid salt thereof or a hydrate thereof isadministered to a patient with lipid-rich plaques. Further, the presentinvention provides an agent for preventing and/or treating acutemyocardial infarction comprising the compound 1, an acid salt thereof ora hydrate thereof as an active ingredient. Further the present inventionprovides a pharmaceutical composition having activities for preventingand/or treating acute myocardial infarction comprising the compound 1,an acid salt thereof or a hydrate thereof, and a pharmaceuticallyacceptable carrier thereof. Still further the present invention providesuse of the compound 1, an acid salt thereof or a hydrate thereof forproduction of the pharmaceutical preparation for preventing and/ortreating acute myocardial infarction.

The present invention provides a method of preventing and/or treatingunstable angina, characterized in that an effective amount of thecompound 1, an acid salt thereof or a hydrate thereof is administered toa patient with lipid-rich plaques. Further, the present inventionprovides an agent for preventing and/or treating unstable anginacomprising the compound 1, an acid salt thereof or a hydrate thereof asan active ingredient. Further the present invention provides apharmaceutical composition having activities for preventing and/ortreating unstable angina comprising the compound 1, an acid salt thereofor a hydrate thereof, and a pharmaceutically acceptable carrier thereof.Still further the present invention provides use of the compound 1, anacid salt thereof or a hydrate thereof for production of thepharmaceutical preparation for preventing and/or treating unstableangina.

The present invention provides a method of preventing and/or treatingperipheral artery obstruction, characterized in that an effective amountof the compound 1, an acid salt thereof or a hydrate thereof isadministered to a patient with lipid-rich plaques. Further, the presentinvention provides an agent for preventing and/or treating peripheralartery obstruction comprising the compound 1, an acid salt thereof or ahydrate thereof as an active ingredient. Further the present inventionprovides a pharmaceutical composition having activities for preventingand/or treating peripheral artery obstruction comprising the compound 1,an acid salt thereof or a hydrate thereof, and a pharmaceuticallyacceptable carrier thereof. Still further the present invention providesuse of the compound 1, an acid salt thereof or a hydrate thereof forproduction of the pharmaceutical preparation for preventing and/ortreating peripheral artery obstruction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows plasma total cholesterol level, when the compound 1 (100mg/kg), CI-1011 (100 mg/kg) or vehicle was administered orally, twice aday for 12 weeks in male ApoE knockout mice.

FIG. 2 shows a cross-sectional area of the plaques measured by stainingthe specimens of surgically removed aortic sinus with Victoria blue-HE(HE: hematoxylin-eosin), after the compound 1 (100 mg/kg), CI-1011 (100mg/kg) or vehicle were administered orally, twice a day for 12 weeks inmale ApoE knockout mice.

FIG. 3 shows photographs showing results of Azan staining of thespecimens of surgically removed aortic sinus, after the compound 1 (100mg/kg), CI-1011 (100 mg/kg) or vehicle were administered orally, twice aday for 12 weeks in male ApoE knockout mice.

FIG. 4 shows photographs showing results of immunohistochemical stainingof macrophages in the specimens of surgically removed aortic sinus,after the compound 1 (100 mg/kg), CI-1011 (100 mg/kg) or vehicle wereadministered orally, twice a day for 12 weeks in male ApoE knockoutmice.

FIG. 5 shows photographs showing results of Sirius Red staining of thespecimens of surgically removed aortic sinus, after the compound 1 (100mg/kg), CI-1011 (100 mg/kg) or vehicle were administered orally, twice aday for 12 weeks in male ApoE knockout mice.

FIG. 6 shows the occupancy rate of macrophage obtained by the imageanalysis of immunohistochemical staining of macrophages in the specimensof surgically removed aortic sinus, after the compound 1 (100 mg/kg),CI-1011 (100 mg/kg) or vehicle were administered orally, twice a day for12 weeks in male ApoE knockout mice.

FIG. 7 shows the occupancy rate of collagen obtained by the imageanalysis of Sirius Red staining of macrophages in the specimens ofsurgically removed aortic sinus, after the compound 1 (100 mg/kg),CI-1011 (100 mg/kg) or vehicle were administered orally, twice a day for12 weeks in male ApoE knockout mice.

BEST MODE FOR CARRYING OUT THE INVENTION

The present inventors have studied effects of administration of ACATinhibitor on the plasma total cholesterol level and the vascular lesionby using ApoE knockout mice.

In the study examining the effect on the plasma total cholesterol level,the compound 1 (100 mg/kg), CI-1011 (100 mg/kg) or vehicle wereadministered orally, twice a day for 12 weeks in male ApoE knockoutmice, and the plasma total cholesterol level was measured. Results areshown in FIG. 1. The axis of ordinate in FIG. 1 indicates the plasmatotal cholesterol level (mg/dl). As shown in FIG. 1, significantlydecreased plasma levels of total cholesterol were shown in any of thecompound 1 administered group and the CI-1011 administered group ascompared with the control group. It was found that the degree of thedecreased value was small in the compound 1, but was remarkable in theCI-1011.

In parallel with the evaluation of the plasma lipids, the aortic sinuswas surgically removed and embedded in paraffin, then serial sectionswere prepared, thereafter Victoria blue-hematoxylin-eosin staining, Azanstaining, macrophage immunohistochemical staining (anti-CD11b antibody)and Sirius Red staining were performed.

Using the specimens of Victoria blue-hematoxylin-eosin staining, theinternal elastic layer was analyzed by observation of opticalmicroscope. The specified cross-sectional area of the plaques in thespecimens is shown in FIG. 2. The axis of ordinate in FIG. 2 indicatesthe cross-sectional area of the plaques (mm²). As shown in FIG. 2, thearea was almost not changed in the compound 1 administered group, butthe cross-sectional area of the plaques was significantly decreased inthe CI-1011 administered group as compared with the control group.

Results of Azan staining are shown in FIG. 3. As shown in FIG. 3, in thecontrol group, the surface layer side of the inner membrane consistslargely of the pale red stained foamy macrophages, and the blue stainedextracellular matrix was slightly observed in the deep layer. Largenumbers of white defective acicular areas called cholesterol craft wereobserved in the plaque. Contrary, although the cross-sectional area ofthe plaque in the compound 1 administered group was not so different ascompared with the control group, the plaque consisted of small area ofcellular components and large numbers of surrounding extracellularmatrix. In CI-1011 administered group, although the cross-sectional areaof the plaque was slightly smaller as compared with that of the controlgroup, the plaque consisted of mostly red stained macrophage and theblue stained extracellular matrix was scanty.

Results of the macrophage immunohistochemical staining are shown in FIG.4. As shown in FIG. 4, although the macrophages were abundantly observedspecifically in the surface layer side of the plaque in the controlgroup, the macrophages were occasionally observed in the compound 1administered group. Contrary, the macrophages were observed abundantlyin whole plaque in the CI-1011 administered group.

Results of Sirius Red staining are shown in FIG. 5. As shown in FIG. 5,compactly-arranged collagen fibers were observed all over the plaque inthe compound 1 administered group as compared with the control group.However, in CI-1011 administered group, smaller numbers of collagenfibers were observed as compared with the control group.

Results of the occupancy rate of macrophage in the plaques calculatedfrom the image analysis in FIG. 4 are shown in FIG. 6. The axis ofordinate in FIG. 6 indicates the occupancy rate (%) of the macrophage inthe plaques. As shown in FIG. 6, the occupancy rate of the macrophagewas significantly decreased in the compound 1 administered group ascompared with the control group. Significant increase was observed inthe CI-1011 administered group.

Results of the occupancy rate of collagen in the plaques calculated fromthe image analysis in FIG. 5 are shown in FIG. 7. The axis of ordinatein FIG. 7 indicates the occupancy rate (%) of the collagen in theplaques. As shown in FIG. 7, the occupancy rate of the collagen wassignificantly increased in the compound 1 administered group as comparedwith the control group. On the other hand, decreasing tendency wasobserved in the CI-1011 administered group.

As described above, it was found that the compound 1 reduced theoccupancy rate of macrophage in the plaques without making significanteffect on changes of the plasma total cholesterol level, and increasedthe occupancy rate of collagen. In this way, the compound 1 increasescollagen level, and simultaneously, reduces the occupancy rate of themacrophage to stabilize the plaques, thereby prevents rupture thereof.

Consequently, the present invention relates to a method of stabilizinglipid-rich plaques and a method of preventing the rupture thereof. Morespecifically, the present invention provides a method of stabilizinglipid-rich plaques and a method of preventing the rupture thereof,characterized in that an effective amount of the pharmaceuticalcomposition containing the active ingredient consisting of one or moretypes of the compound 1, an acid salt thereof or a hydrate thereof isadministered to a patient with lipid-rich plaques. Further, the presentinvention provides a pharmaceutical composition having activities forstabilizing lipid-rich plaques and preventing the rupture thereofcomprising the active ingredient consisting of one or more types of thecompound 1, an acid salt thereof or a hydrate thereof, and apharmaceutically acceptable carrier thereof.

Since the compound 1, an acid salt thereof or a hydrate thereof of thepresent invention increases collagen in the lipid-rich plaques andreduces the occupancy rate of the macrophage, it can stabilize thelipid-rich plaques and prevents rupture thereof, thereby preventsthrombogenesis. Consequently, the present invention provides thepreventive agents and/or therapeutic agents of various thrombus relateddiseases such as thrombosis caused by thrombogenesis, acute coronarysyndrome, acute myocardial infarction, unstable angina, and peripheralartery obstruction, the pharmaceutical composition thereof, method forprevention and/or treatment using the same, and use for productionthereof.

The pharmaceutical composition having stabilizing activity for plaquesof the present invention comprises the compound 1 of the presentinvention as an active ingredient combined with a pharmaceuticallyacceptable carrier in the composition appropriate for administrationform such as oral form or parenteral form, for example oraladministration form, injectable form, suppository, ointment and patch,and can be produced by the formulation method known by the personskilled in the art. Also, in the present invention, an acid salt of thecompound 1 or a hydrate of the compound 1 or an acid salt of thecompound 1 can be used. The acid salt and the hydrate can be produced bythe conventional method. Examples of acid for forming acid salt hereinare inorganic acid such as hydrochloric acid, sulfuric acid, nitricacid, phosphoric acid, hydrobromic acid and hydroiodic acid, and organicacid such as acetic acid, lactic acid, succinic acid, tartaric acid,malic acid, maleic acid, fumaric acid, citric acid, ascorbic acid,methanesulfonic acid, besilate and toluenesulfonic acid. The compound 1,an acid salt thereof or a hydrate thereof can be used alone as an activeingredient, but can also be used in the mixture of two or more kinds asthe active ingredient.

In the production of solid preparation for oral administration, forexample, tablets, coated tablets, granules, powders and capsules can beproduced by the conventional manner after diluent, if necessary, binder,disintegrator, lubricant, coloring agent, flavoring substance andcorrigent are added to the compound 1, an acid salt thereof or a hydratethereof. Examples of such additives can be the commonly used materialsin the relevant field. For example, diluent include lactose, white softsugar, sodium chloride, glucose, starch, calcium carbonate, kaolin,crystalline cellulose, silicic acid, etc.; binders include water,ethanol, propanol, simple syrup, glucose solution, starch suspension,gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose,hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calciumphosphate, polyvinylpyrrolidone, etc.; disintegrators include drystarch, sodium alginate, agar powder, sodium hydrogen carbonate, calciumcarbonate, sodium lauryl sulfate, mono glyceride stearate, lactose,etc.; lubricants include purified talc, stearate, sodium borate,polyethylene glycol, etc.; and corrigents such as white soft sugar,bitter orange peel, citric acid, tartaric acid, etc.

In case of preparing liquids and solutions for oral administration, forexample, the liquid preparation for oral administration, syrup, elixir,etc. can be produced in the conventional means by adding, if necessary,flavoring substance, buffer, stabilizing agent, corrigent, etc. to theabove described compound 1, an acid salt thereof or a hydrate thereof.Examples of flavoring substances are as mentioned above, and those ofbuffers are sodium citrate, etc. and stabilizing agents are tragacanth,gum acacia, gelatin, etc.

In case of preparing injectable preparation, for example, thesubcutaneous injection, intramuscular injection, intravenous injection,etc. can be produced in the conventional means by adding, if necessary,pH adjusting agent, buffer, stabilizing agent, tonicity adjusting agent,local anesthetic, etc. to the above described compound 1, an acid saltthereof or a hydrate thereof. Examples of the pH adjusting agent andbuffer are sodium citrate, sodium acetate, sodium phosphate, etc.Examples of stabilizing agent are sodium pyrosulfite, EDTA, thioglycolicacid, thiolactic acid, etc. Examples of local anesthetics are procainehydrochloride, lidocaine hydrochloride, etc. Examples of tonicityadjusting agents are sodium chloride, glucose, etc.

Other dosage forms can be produced according to the known methods. Thethus obtained pharmaceutical composition having activity for stabilizingplaques of the present invention is effective for stabilizing theplaques in atherosclerotic lesions, prevention of thrombogenesisaccompanied to rupture of plaques, prevention and/or treatment of acutecoronary syndrome, prevention and/or treatment of acute myocardialinfarction, prevention and/or treatment of unstable angina, andprevention and/or treatment of peripheral artery obstruction.

An applied dose of the pharmaceutical composition having activity forstabilizing plaques of the present invention depends on body weight,age, sex, symptoms of patients, dosage form and frequency ofadministration, and is preferably administered generally the compound 1,an acid salt thereof or a hydrate thereof, 0.01-1000 mg/day, preferably0.1-100 mg/day, in adult, once or several times a day by oral orparenteral administration.

The contents described in U.S. Patent Application No. 60/498,610 areincorporated herein in the entirety thereof.

The present invention will be explained in detail by way of Exampleshereinbelow, but the present invention is not limited thereto.

Example 1

Anti-arterial sclerotic action of the drug was examined according to themethod described herein below by using ApoE knockout mice with primaryhyperlipidemia.

(1) Test Method 1. Test Animal

Home breeding male ApoE knockout mice, (C57BL/6J-ApoE<tmlUnc>, JacksonLabo™) were bred under common condition, 8 weeks old, were used fortest.

2. Test Drug and Preparation of the Test Drug, and Method and Term ofAdministration

The compound 1 (Example 32, WO98/54153) and control drug, CI-1011(Example 5, WO 94/26702), were synthesized according to methodsdescribed in each publication. The compound 1 and CI-1011 were dissolved(the compound 1) or suspended (CI-1011) in aqueous solution of 0.5%methylcellulose (MC). The solution or suspension was prepared to be adose of 100 mg/kg and a volume for administration of 0.1 mL/10 g bodyweight. The control group (no drug administered group) received thesolvent, aqueous solution of 0.5% methylcellulose (MC). Prepared drugsolution and suspension were administered twice a day, orally, forcontinuing to 12 weeks (up to 20 weeks old) (total 3 groups, each 15animal).

3. Observational and Inspection Method A. Plasma Total Cholesterol Level

After ending the final administration, mice were fasted. In the nextmorning, mice were laparotomized under pentobarbital sodium anesthesiato expose abdominal vein and collected blood sample 1 mL. The collectedblood was centrifuged at 3000 rpm for 15 minutes to collect the plasma.Plasma total cholesterol level was assayed by using cholesterol E-testWako (cholesterol oxidase DAOS method).

B. Histological Evaluation

After collecting the blood, animals were undergone thoracotomy and 20-Ginjection needle was punctured into the cardiac apex. Physiologicalsaline and subsequently 4% paraformaldehyde were perfused from thecardiac apex under the perfusion pressure at 120 cmH₂O for 5 minutes tofixate by perfusion. Heart and thoracic aorta were collected and fixatedwith the fixative by immersing for a day or more. Thereafter, aorticsinus was cut off and embedded in paraffin. Serial sections of specimenswere prepared and stained with Victoria blue-hematoxylin and eosinstaining, Azan staining, macrophage immunohistochemical staining(anti-CD11b antibody) and Sirius Red staining. Using the specimens ofVictoria blue-hematoxylin and eosin staining, the internal elastic layerwas specified by observation of optical microscope, and thecross-sectional area of the plaques was analyzed (Win ROOF, MitsuyaShoji K.K.). Similarly, the area of macrophages was measured by usingthe specimens of macrophage immunohistochemical staining, and a ratio intotal area of the plaques (the occupancy rate of macrophages) wascalculated. Further, the area of collagen was measured by using SiriusRed staining, and a ratio occupied with collagen in total area of theplaques (the occupancy rate of collagen) was calculated.

4. Statistical Analysis and Data Processing

Obtained results are shown in mean±standard error. Test of significancebetween the control group and the drug-administered group is performedby Dunnett's test.

(2) Results A. Plasma Total Cholesterol Level

Plasma total cholesterol level in each group is shown in FIG. 1. Theplasma total cholesterol level in the control group is 641.8±23.0 mg/dL,whereas the plasma total cholesterol level in the compound 1, 100 mg/kgadministered group is 535.0±22.8 mg/dL, and although it is significant(P<0.01), low decreasing effect is shown. The plasma total cholesterollevel in the CI-1011, 100 mg/kg administered group is 360.3±19.4 mg/dL,and it is significant (P<0.01), and is characterized by a great loweringeffect as compared with the compound 1. As the results of the above,tested drugs exhibit decreased action for plasma total cholesterollevel, and the activity is found to exhibit remarkably in CI-1011, 100mg/kg administered group.

B. Histological Evaluation

The cross-sectional area of the plaques measured by Victoriablue-hematoxylin and eosin staining in each group is shown in FIG. 2.The cross-sectional area of the plaques in the control group is0.23±0.03 mm², whereas the cross-sectional area of the plaques in thecompound 1, 100 mg/kg administered group, is 0.22±0.03 mm², consequentlyno difference was observed. Contrary to that, the cross-sectional areaof the plaques in the CI-1011, 100 mg/kg administered group, is0.09±0.01 mm², consequently reducing action for the cross-sectional areaof the plaques is stronger than the compound 1 and is exhibitedsignificantly (P<0.01). As the results of the above, the reducing actionfor the cross-sectional area of the plaques, namely the plaque reducingaction is observed in the CI-1011, 100 mg/kg administered group.

Results of Azan staining, macrophage immunohistochemical staining andSirius Red staining are shown in FIG. 3-FIG. 5. In each figure, theunder side corresponds to the adventitia.

In the specimens of Azan staining (FIG. 3), the surface layer side ofthe intima consists largely of the pale red stained foamy macrophages,and the blue stained extracellular matrix was slightly observed in thedeep layer. Large numbers of white defective acicular areas calledcholesterol craft were observed. Contrary to that, although thecross-sectional area of the plaques in the compound 1, 100 mg/kgadministered group was not so different as compared with the controlgroup, the plaques consisted of small area of cellular components andlarge numbers of surrounding extracellular matrix. In CI-1011administered group, although the cross-sectional area of the plaques wassmaller as compared with that of the control group, the plaquesconsisted largely of red stained macrophage and the blue stainedextracellular matrix was scanty.

Results of the macrophage immunohistochemical staining are shown in FIG.4. As shown in FIG. 4, although the macrophages were abundantly observedspecifically in the surface layer side in the control group, themacrophages were occasionally observed in the compound 1 administeredgroup. Contrary to that, the macrophages were observed abundantly inwhole plaques in the CI-1011 administered group.

Results of Sirius Red staining are shown in FIG. 5. As shown in FIG. 5,compactly arranged collagen fibers were observed in whole plaques in thecompound 1 administered group as compared with the control group.However, in CI-1011 administered group, smaller numbers of collagenfibers were observed as compared with the control group.

Results of the occupancy rate of macrophage in the plaques calculatedfrom the image analysis in FIG. 4 are shown in FIG. 6. As shown in FIG.6, the occupancy rate of the macrophage was 33.2±2.2% in the controlgroup, whereas the compound 1, 100 mg/kg administered group wassignificantly decreased to 20.0±1.7% (P<0.01). Contrary to that, it wassignificantly increased to 44.0±2.7% (P<0.01) in the CI-1011, 100 mg/kgadministered group.

Results of the occupancy rate of collagen in the plaques calculated fromthe image analysis in FIG. 5 are shown in FIG. 7. As shown in FIG. 7,the occupancy rate of the collagen was 9.6±1.1% in the control group,whereas it was significantly increased to 14.9±1.0% in the compound 1,100 mg/kg administered group (P<0.05). On the other hand, decreasingtendency was shown as 7.0±0.9% in the CI-1011, 100 mg/kg administeredgroup.

As obvious from FIG. 6 and FIG. 7, the significant decrease of theoccupancy rate of macrophage and the significant increase of theoccupancy rate of collagen were observed in the compound 1, 100 mg/kgadministered group as compared with the control group, whereas thesignificant increase of the occupancy rate of macrophage and decreasedtendency of the occupancy rate of collagen were observed in the CI-1011,100 mg/kg administered group. Namely, quite unexpectedly, even in thesimilar ACAT inhibitor, completely different action was confirmedagainst the plaques.

As explained hereinabove, effects of the compound 1 and CI-1011 havingreducing activity for the plasma total cholesterol level based on ACATinhibitory action were examined on the plasma total cholesterol level,the cross-sectional area of the plaques, and the histologicalevaluation. Results indicated that the CI-1011 exhibited decreasingeffect on the cross-sectional area of the plaques, i.e. the plaquereducing action, at the concentration for decreasing the plasma totalcholesterol level, but exhibited increase in the occupancy rate of themacrophage and decrease in the occupancy rate of the collagen,consequently it was demonstrated that the CI-1011 rather destabilizedthe plaques qualitatively. On the contrary, the compound 1 only slightlyreduced the plasma total cholesterol level and exhibited no plaquereducing action, but exhibited decrease in the occupancy rate of themacrophage and increase in the occupancy rate of the collagen,consequently it was demonstrated that the compound 1 had remarkableplaque stabilizing action.

INDUSTRIAL APPLICABILITY

According to the present invention, a method of stabilizing lipid-richplaques in atherosclerotic lesions and a method of preventing therupture thereof can be provided. More specifically, a method ofstabilizing lipid-rich plaques and a method of preventing the rupturethereof by suppressing accumulation of macrophages and increasingcollagen in the plaque lesions by administering an effective amount of2-[4-[2-(benzimidazol-2-ylthio)ethyl]piperazin-1-yl]-N-[2,4-bis(methylthio)-6-methyl-3-pyridyl]acetamide,an acid salt thereof or a hydrate thereof can be provided.

1. A method of preventing acute coronary syndrome due to the rupture oflipid-rich plaques, comprising administering a therapeutically effectiveamount of2-[4-[2-(benzimidazol-2-ylthio)ethyl]piperazin-1-yl]-N-[2,4-bis(methylthio)-6-methyl-3-pyridyl]acetamide,an acid salt thereof or a hydrate thereof to a patient with lipid-richplaques, whereby rupture of lipid rich plaques is prevented.
 2. A methodof preventing acute myocardial infarction due to the rupture oflipid-rich plaques, comprising administering a therapeutically effectiveamount of2-[4-[2-(benzimidazol-2-ylthio)ethyl]piperazin-1-yl]-N-[2,4-bis(methylthio)-6-methyl-3-pyridyl]acetamide,an acid salt thereof or a hydrate thereof to a patient with lipid-richplaques, whereby rupture of lipid rich plaques is prevented.
 3. A methodof preventing unstable angina due to the rupture of lipid-rich plaques,comprising administering a therapeutically effective amount of2-[4-[2-(benzimidazol-2-ylthio)ethyl]piperazin-1-yl]-N-[2,4-bis(methylthio)-6-methyl-3-pyridyl]acetamide,an acid salt thereof or a hydrate thereof to a patient with lipid-richplaques, whereby rupture of lipid rich plaques is prevented.
 4. A methodof preventing peripheral artery obstruction due to the rupture oflipid-rich plaques, comprising administering a therapeutically effectiveamount of2-[4-[2-(benzimidazol-2-ylthio)ethyl]piperazin-1-yl]-N-[2,4-bis(methylthio)-6-methyl-3-pyridyl]acetamide,an acid salt thereof or a hydrate thereof to a patient with lipid-richplaques, whereby rupture of lipid rich plaques is prevented.